Nucleotide sequence which code for the metH gene

ABSTRACT

An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) polynucleotide which is at least 70% identical to a polynucleotide that codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, b) polynucleotide which codes for a polypeptide that comprises an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID No. 2, c) polynucleotide which is complementary to the polynucleotides of a) or b), and d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of a), b) or c), and processes for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the metH gene is present in enhanced form, and use of the polynucleotide sequences as hybridization probes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention provides nucleotide sequences from coryneform bacteriawhich code for the metH gene and a process for the fermentativepreparation of amino acids, in particular L-methionine, using bacteriain which the metH gene is enhanced.

2. Description of the Related Art

L-Amino acids, in particular L-methionine, are used in human medicineand in the pharmaceuticals industry, in the foodstuffs industry and veryparticularly in animal nutrition.

It is known that amino acids are prepared by fermentation from strainsof coryneform bacteria, in particular Corynebacterium glutamicum.Because of their great importance, work is constantly being undertakento improve the preparation process. Improvements to the process canrelate to fermentation measures, such as, stirring and supply of oxygen,or to the composition of the nutrient media, such as, the sugarconcentration during the fermentation, or to the working up of theproduct by, for example, ion exchange chromatography, or to theintrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used toimprove the output properties of these microorganisms. Strains which areresistant to antimetabolites, such as e.g. the methionine analogueα-methyl-methionine, ethionine, norleucine, N-acetylnorleucine,S-trifluoromethylhombcysteine, 2-amino-5-heprenoitic acid,seleno-methionine, methionine-sulfoximine, methoxine,1-aminocyclopentane-carboxylic acid, or are auxotrophic for metabolitesof regulatory importance and produce amino acids, such as e.g.L-methionine, are obtained in this manner.

Recombinant DNA techniques have also been employed for some years forimproving Corynebacterium strains which produce L-amino acids, byamplifying individual amino acid biosynthesis genes and investigatingtheir effect on the amino acid production.

SUMMARY OF THE INVENTION

One object of the present invention is to provide new measures forimproved fermentative preparation of amino acids, in particularL-methionine.

When L-methionine or methionine are mentioned in the following, thesalts, such as methionine hydrochloride or methionine sulfate are alsomeant.

The invention provides an isolated polynucleotide from coryneformbacteria, comprising a polynucleotide sequence which codes for the metHgene, chosen from the group consisting of

-   -   a) polynucleotide which is at least 70% identical to a        polynucleotide that codes for a polypeptide which comprises the        amino acid sequence of SEQ ID No. 2,    -   b) polynucleotide which codes for a polypeptide that comprises        an amino acid sequence which is at least 70% identical to the        amino acid sequence of SEQ ID No. 2,    -   c) polynucleotide which is complementary to the polynucleotides        of a) or b), and    -   d) polynucleotide comprising at least 15 successive nucleotides        of the polynucleotide sequence of a), b) or c), and the        corresponding polypeptides having the enzymatic activity of        homocysteine methyltransferase II.

The invention also provides the above-mentioned polynucleotides, as DNAwhich is capable of replication, comprising:

-   -   (i) the nucleotide sequence shown in SEQ ID No. 1, or    -   (ii) at least one sequence which corresponds to sequence (i)        within the range of the degeneration of the genetic code, or    -   (iii) at least one sequence which hybridizes with the sequence        complementary to sequence (i) or (ii), and optionally    -   (iv) sense mutations of neutral function in (i).

The invention also provides

-   -   a polynucleotide comprising the nucleotide sequence as shown in        SEQ ID No. 1;    -   a polynucleotide that codes for a polypeptide which comprises        the amino acid sequence as shown in SEQ ID No. 2,    -   a vector containing the polynucleotide according to the        invention, in particular a shuttle vector or plasmid vector, and    -   and coryneform bacteria serving as the host cell, which contain        the vector or in which the metH gene is enhanced.

The invention also provides polynucleotides which are obtained byscreening a corresponding gene library, which comprises the completegene having the polynucleotide sequence corresponding to SEQ ID No. 1,by means of hybridization with a probe which comprises the sequence ofthe polynucleotide mentioned, according to SEQ ID No. 1 or a fragmentthereof, and isolation of the DNA sequence mentioned.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows plasmid pCREmetH.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Polynucleotides according to the invention are suitable as hybridizationprobes for RNA, cDNA and DNA, in order to isolate, in the full length,nucleic acids or polynucleotides or genes which code for homocysteinemethyltransferase II or to isolate those nucleic acids orpolynucleotides or genes which have a high similarity of sequence orhomology with that of the homocysteine methyltransferase II gene.

Polynucleotides according to the invention are furthermore suitable asprimers with the aid of which DNA of genes that code for homocysteinemethyltransferase II can be prepared by the polymerase chain reaction(PCR).

Such oligonucleotides that serve as probes or primers comprise at least30, preferably at least 20, very particularly at least 15 successivenucleotides. oligonucleotides which have a length of at least 40 or 50nucleotides are also suitable. Oligonucleotides with a length of atleast 100, 150, 200, 250 or 300 nucleotides are optionally alsosuitable.

“Isolated” means separated out of its natural environment.

“Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

“Polypeptides” are understood as meaning peptides or proteins whichcomprise two or more amino acids bonded via peptide bonds.

The polypeptides according to the invention include a polypeptideaccording to SEQ ID No. 2, in particular those with the biologicalactivity of homocysteine methyltransferase II, and also those which areat least 70%, preferably at least 80% and in particular which are atleast 90% to 95% identical to the polypeptide according to SEQ ID No. 2and have the activity mentioned.

The invention moreover provides a process for the fermentativepreparation of amino acids, in particular L-methionine, using coryneformbacteria which in particular already produce amino acids, and in whichthe nucleotide sequences which code for the metH gene are enhanced, inparticular over-expressed.

The term “enhancement” in this connection describes the increase in theintracellular activity of one or more enzymes (proteins) in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or using a gene or allele which codes for a correspondingenzyme (protein) having a high activity, and optionally combining thesemeasures.

By enhancement measures, in particular over-expression, the activity orconcentration of the corresponding protein is in general increased by atleast 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to amaximum of 1000% or 2000%, based on the starting microorganism.

The microorganisms which the present invention provides can prepareL-amino acids, in particular L-methionine, from glucose, sucrose,lactose, fructose, maltose, molasses, starch, cellulose or from glyceroland ethanol. They can be representatives of coryneform bacteria, inparticular of the genus Corynebacterium. Of the genus Corynebacterium,there may be mentioned in particular the species Corynebacteriumglutamicum, which is known among experts for its ability to produceL-amino acids.

Suitable strains of the genus Corynebacterium, in particular of thespecies Corynebacterium glutamicum (C. glutamicum), are in particularthe known wild-type strains

-   -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium acetoglutamicum ATCC15806    -   Corynebacterium acetoacidophilum ATCC13870    -   Corynebacterium thermoaminogenes FERM BP-1539    -   Corynebacterium melassecola ATCC17965    -   Brevibacterium flavum ATCC14067    -   Brevibacterium lactofermentum ATCC13869 and    -   Brevibacterium divaricatum ATCC14020        or L-amino acid-producing mutants or strains prepared therefrom,        such as, for example, the L-methionine-producing strain    -   Corynebacterium glutamicum ATCC21608.

The new metH gene from C. glutamicum which codes for the enzymehomocysteine methyltransferase II (EC 2.1.1.13) has been isolated.

To isolate the metH gene or also other genes of C. glutamicum, a genelibrary of this microorganism is first set up in Escherichia coli (E.coli). The setting up of gene libraries is described in generally knowntextbooks and handbooks. The textbook by Winnacker: Gene und Klone, EineEinführung in die Gentechnologie (Verlag Chemie, Weinheim, Germany,1990), or the handbook by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may bementioned as examples. A well-known gene library is that of the E. coliK-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50, 495-508(1987)). Bathe et al. (Molecular and General Genetics, 252:255-265,1996) describe a gene library of C. glutamicum ATCC13032, which was setup with the aid of the cosmid vector SuperCos I (Wahl et al., 1987,Proceedings of the National Academy of Sciences USA, 84:2160-2164) inthe E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic AcidsResearch 16:1563-1575).

Börmann et al. (Molecular Microbiology 6(3), 317-326) (1992)) in turndescribe a gene library of C. glutamicum ATCC13032 using the cosmidpHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). To prepare a genelibrary of C. glutamicum in E. coli it is also possible to use plasmidssuch as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9(Vieira et al., 1982, Gene, 19:259-268). Suitable hosts are, inparticular, those E. coli strains which are restriction- andrecombination-defective. An example of these is the strain DH5αmcr,which has been described by Grant et al. (Proceedings of the NationalAcademy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragmentscloned with the aid of cosmids can in turn be subcloned in the usualvectors suitable for sequencing and then sequenced, as is described e.g.by Sanger et al. (Proceedings of the National Academy of Sciences of theUnited States of America, 74:5463-5467, 1977).

The resulting DNA sequences can then be investigated with knownalgorithms or sequence analysis programs, such as that of Staden(Nucleic Acids Research 14, 217-232(1986)), that of Marck (Nucleic AcidsResearch 16, 1829-1836 (1988)) or the GCG program of Butler (Methods ofBiochemical Analysis 39, 74-97 (1998)).

The new DNA sequence of C. glutamicum which codes for the metH gene andwhich, as SEQ ID No. 1, is a constituent of the present invention hasbeen found. The amino acid sequence of the corresponding protein hasfurthermore been derived from the present DNA sequence by the methodsdescribed above. The resulting amino acid sequence of the metH geneproduct is shown in SEQ ID No. 2.

Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy ofthe genetic code are also a constituent of the invention. In the sameway, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ IDNo. 1 are a constituent of the invention. Conservative amino acidexchanges, such as e.g. exchange of glycine for alanine or of asparticacid for glutamic acid in proteins, are furthermore known among expertsas “sense mutations” which do not lead to a fundamental change in theactivity of the protein, i.e. they are of neutral function.

It is furthermore known that changes at the N and/or C terminus of aprotein must not substantially impair and may even stabilize thefunction thereof. Information in this context can be found in Ben-Bassatet al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al.(Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988))and in known textbooks of genetics and molecular biology. Amino acidsequences which result in a corresponding manner from SEQ ID No. 2 arealso a constituent of the invention.

In the same way, DNA sequences which hybridize with SEQ ID No. 1 orparts of SEQ ID No. 1 are a constituent of the invention. Finally, DNAsequences which are prepared by the polymerase chain reaction (PCR)using primers which result from SEQ ID No. 1 are a constituent of theinvention. Such oligonucleotides typically have a length of at least 15nucleotides.

Instructions for identifying DNA sequences by means of hybridization canbe found in the handbook “The DIG System Users Guide for FilterHybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993)and in Liebl et al. (International Journal of Systematic Bacteriology(1991) 41: 255-260). Instructions for amplification of DNA sequenceswith the aid of the polymerase chain reaction (PCR) can be found in thehandbook by Gait: Oligonucleotide Synthesis: A Practical Approach (IRLPress, Oxford, UK, 1984) and in Newton and Graham: PCR (SpektrumAkademischer verlag, Heidelberg, Germany, 1994).

It has been found that coryneform bacteria produce amino acids, inparticular L-methionine, in an improved manner after over-expression ofthe metH gene.

To achieve an over-expression, the number of copies of the correspondinggenes can be increased, or the promoter and regulation region or theribosome binding site upstream of the structural gene can be mutated.Expression cassettes which are incorporated upstream of the structuralgene act in the same way. By inducible promoters, it is additionallypossible to increase the expression in the course of fermentativeL-methionine production. The expression is likewise improved by measuresto prolong the life of the m-RNA. Furthermore, the enzyme activity isalso increased by preventing the degradation of the enzyme protein. Thegenes or gene constructs can either be present in plasmids with avarying number of copies, or can be integrated and amplified in thechromosome. Alternatively, an over-expression of the genes in questioncan furthermore be achieved by changing the composition of the media andthe culture procedure.

Instructions in this context can be found in Martin et al.(Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41(1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), inEikmanns et al. (Gene 102, 93-98 (1991)), in European PatentSpecification 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer andPuhler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Appliedand Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al.(Journal of Bacteriology 175, 1001-1007 (1993)), in Patent ApplicationWO 96/15246, in Malumbres et al. (Gene 134, 15- 24 (1993)), in JapaneseLaid-Open Specification JP-A-10-229891, in Jensen and Hammer(Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides(Microbiological Reviews 60:512-538 (1996)) and in known textbooks ofgenetics and molecular biology.

By way of example, for enhancement the metH gene according to theinvention was over-expressed with the aid of episomal plasmids. Suitableplasmids are those which are replicated in coryneform bacteria. Numerousknown plasmid vectors, such as e.g. pZl (Menkel et al., Applied andEnvironmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al.,Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991))are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmidvectors, such as those based on pCG4 (U.S. Pat. No. 4,489,160), or pNG2(Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), orpAG1 (U.S. Pat. No. 5,158,891), can be used in the same manner. Plasmidvectors which are furthermore suitable are also those with the aid ofwhich the process of gene amplification by integration into thechromosome can be used, as has been described, for example, byReinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)) for duplication or amplification of the hom-thrB operon. In thismethod, the complete gene is cloned in a plasmid vector which canreplicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schafer et al., Gene 145, 69-73(1994)), pGEM-T (Promega corporation, Madison, Wis., U.S.A.),pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry269:32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen,Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)), pEM1 (Schrumpf et al, 1991, Journal of Bacteriology173:4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342). Theplasmid vector which contains the gene to be amplified is thentransferred into the desired strain of C. glutamicum by conjugation ortransformation. The method of conjugation is described, for example, bySchafer et al. (Applied and Environmental Microbiology 60, 756-759(1994)). Methods for transformation are described, for example, byThierbach et al. (Applied Microbiology and Biotechnology 29, 356-362(1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) andTauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). Afterhomologous recombination by means of a “cross over” event, the resultingstrain contains at least two copies of the gene in question.

In addition, it may be advantageous for the production of amino acids,in particular L-methionine, to enhance one or more enzymes of theparticular biosynthesis pathway, of glycolysis, of anaplerosis, of thecitric acid cycle or of amino acid export, in addition to the metH gene.

Thus for the preparation of amino acids, in particular L-methionine, oneor more genes chosen from the group consisting of

-   -   the gap gene which codes for glyceraldehyde 3-phosphate        dehydrogenase (Eikmanns (1992), Journal of Bacteriology        174:6076-6086),    -   the tpi gene which codes for triose phosphate isomerase        (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),    -   the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns        (1992), Journal of Bacteriology 174:6076-6086),    -   the pyc gene which codes for pyruvate carboxylase (Eikmanns        (1992), Journal of Bacteriology 174:6076-6086),    -   the lysC gene which codes for a feed-back resistant aspartate        kinase (ACCESSION NUMBER P26512; EP-B-0387527; EP-A-0699759),    -   the metA gene which codes for homoserine O-acetyltransferase        (ACCESSION Number AF052652),    -   the metB gene which codes for cystathionine gamma-synthase        (ACCESSION Number AF126953),    -   the aecD gene which codes for cystathionine gamma-lyase        (ACCESSION Number M89931)    -   the glyA gene which codes for serine hydroxymethyltransferase        (JP-A-08107788),    -   the metY gene which codes for O-acetylhomoserine-sulfhydrylase        (DSM 13556)        can be enhanced, in particular over-expressed.

It may furthermore be advantageous for the production of amino acids, inparticular L-methionine, in addition to the enhancement of the metHgene, for one or more genes chosen from the group consisting of

-   -   thrB gene which codes for homoserine kinase (ACCESSION Number        P08210),    -   the ilvA gene which codes for threonine dehydratase (ACCESSION        Number Q04513),    -   the thrC gene which codes for threonine synthase (ACCESSION        Number P23669),    -   the ddh gene which codes for meso-diaminopimelate        D-dehydrogenase (ACCESSION Number Y00151),    -   the pck gene which codes for phosphoenol pyruvate carboxykinase        (DE 199 50 409.1; DSM 13047),    -   the pgi gene which codes for glucose 6-phosphate isomerase (U.S.        Pat. No. 09/396,478; DSM 12969),    -   the poxB gene which codes for pyruvate oxidase (DE: 1995 1975.7;        DSM 13114)        to be attenuated, in particular for the expression thereof to be        reduced.

The term “attenuation” in this connection describes the reduction orelimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or using a gene or allele whichcodes for a corresponding enzyme with a low activity or inactivates thecorresponding gene or enzyme (protein), and optionally combining thesemeasures.

By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 50%, 0 to 25%, 0 to10% or 0 to 5% of the activity or concentration of the wild-typeprotein.

In addition to over-expression of the metH gene it may furthermore beadvantageous for the production of amino acids, in particularL-methionine, to eliminate undesirable side reactions, (Nakayama:“Breeding of Amino Acid Producing Micro-organisms”, in: Overproductionof Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press,London, UK, 1982).

The microorganisms prepared according to the invention can be culturedcontinuously or discontinuously in the batch process (batch culture) orin the fed batch (feed process) or repeated fed batch process(repetitive feed process) for the purpose of production of amino acids,in particular L-methionine. A summary of known culture methods isdescribed in the textbook by Chmiel (Bioprozesstechnik 1. Einführung indie Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen(Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose,fructose, maltose, molasses, starch and cellulose, oils and fats, suchas e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fattyacids, such as e.g. palmitic acid, stearic acid and linoleic acid,alcohols, such as e.g. glycerol and ethanol, and organic acids, such ase.g. acetic acid, can be used as the source of carbon. These substancescan be used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract,meat extract, malt extract, corn steep liquor, soya bean flour and urea,or inorganic compounds, such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, can be usedas the source of nitrogen. The sources of nitrogen can be usedindividually or as a mixture.

Organic and inorganic sulfur-containing compounds, such as, for example,sulfides, sulfites, sulfates and thiosulfates, can be used as a sourceof sulfur, in particular for the preparation of methionine.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used asthe source of phosphorus. The culture medium must furthermore comprisesalts of metals, such as e. g. magnesium sulfate or iron sulfate, whichare necessary for growth. Finally, essential growth substances, such asamino acids and vitamins, can be employed in addition to theabove-mentioned substances. Suitable precursors can moreover be added tothe culture medium. The starting substances mentioned can be added tothe culture in the form of a single batch, or can be fed in during theculture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acid compounds, such as phosphoric acid orsulfuric acid, can be employed in a suitable manner to control the pH ofthe culture. Antifoams, such as e.g. fatty acid polyglycol esters, canbe employed to control the development of foam. Suitable substanceshaving a selective action, such as e.g. antibiotics, can be added to themedium to maintain the stability of plasmids. To maintain aerobicconditions, oxygen or oxygen-containing gas mixtures, such as e.g. air,are introduced into the culture. The temperature of the culture isusually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing iscontinued until a maximum of the desired product has formed. This targetis usually reached within 10 hours to 160 hours.

The fermentation broths obtained in this way, in particular containingL-methionine, usually have a dry weight of 7.5 to 25 wt. % and containL-methionine. It is furthermore also advantageous if the fermentation isconducted in a sugar-limited procedure at least at the end, but inparticular over at least 30% of the duration of the fermentation. Thatis to say, the concentration of utilizable sugar in the fermentationmedium is reduced to ≧0 to 3 g/l during this period.

The fermentation broth prepared in this manner, in particular containingL-methionine, is then further processed. Depending on requirements allor some of the biomass can be removed from the fermentation broth byseparation methods, such as centrifugation, filtration, decanting or acombination thereof, or it can be left completely in. This broth is thenthickened or concentrated by known methods, such as with the aid of arotary evaporator, thin film evaporator, falling film evaporator, byreverse osmosis, or by nanofiltration. This concentrated fermentationbroth can then be worked up by methods of freeze drying, spray drying,spray granulation or by other processes to give a preferablyfree-flowing, finely divided powder.

This free-flowing, finely divided powder can then in turn by convertedby suitable compacting or granulating processes into a coarse-grained,readily free-flowing, storable and largely dust-free product. In thegranulation or compacting it is advantageous to employ conventionalorganic or inorganic auxiliary substances or carriers, such as starch,gelatin, cellulose derivatives or similar substances, such as areconventionally used as binders, gelling agents or thickeners infoodstuffs or feedstuffs processing, or further substances, such as, forexample, silicas, silicates or stearates.

“Free-flowing” is understood as meaning powders which flow unimpeded outof the vessel with the opening of 5 mm (millimeters) of a series ofglass outflow vessels with outflow openings of various sizes (Klein,Seifen, Öle, Fette, Wachse 94, 12 (1968)).

As described here, “finely divided” means a powder with a predominantcontent (>50%) having a particle size of 20 to 200 μm diameter.“Coarse-grained” means products with a predominant content (>50%) havinga particle size of 200 to 2000 μm diameter. In this context, “dust-free”means that the product contains only small contents (<5%) havingparticle sizes of less than 20 μm diameter. The particle sizedetermination can be carried out with methods of laser diffractionspectrometry. The corresponding methods are described in the textbook on“Teilchengröβenmessung in der Laborpraxis” by R. H. Müller and R.Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) or inthe textbook “Introduction to Particle Technology” by M. Rhodes, VerlagWiley & Sons (1998).

“Storable” in the context of this invention means a product which can bestored for up to 120 days, preferably up to 52 weeks, particularlypreferably 60 months, without a substantial loss (<5%) of methionineoccurring.

Alternatively, however, the product can be absorbed on to an organic orinorganic carrier substance which is known and conventional infeedstuffs processing, for example, silicas, silicates, grits, brans,meals, starches, sugars or others, and/or mixed and stabilized withconventional thickeners or binders. Use examples and processes in thiscontext are described in the literature (Die Mühle+Mischfuttertechnik132 (1995) 49, page 817).

Finally, the product can be brought into a state in which it is stableto digestion by animal stomachs, in particular the stomach of ruminants,by coating processes (“coating”) using film-forming agents, such as, forexample, metal carbonates, silicas, silicates, alginates, stearates,starches, gums and cellulose ethers, as described in DE-C-4100920.

If the biomass is separated off during the process, further inorganicsolids, for example added during the fermentation, are in generalremoved. In addition,. the animal feedstuffs additive according to theinvention comprises at least the predominant proportion of the furthersubstances, in particular organic substances, which are formed or addedand are present in solution in the fermentation broth, where these havenot been separated off by suitable processes.

In one aspect of the invention, the biomass can be separated off to theextent of up to 70%, preferably up to 80%, preferably up to 90%,preferably up to 95%, and particularly preferably up to 100%. In anotheraspect of the invention, up to 20% of the biomass, preferably up to 15%,preferably up to 10%, preferably up to 5%, particularly preferably nobiomass is separated off.

These organic substances include organic by-products which areoptionally produced, in addition to the L-methionine, and optionallydischarged by the microorganisms employed in the fermentation. Theseinclude L-amino acids chosen from the group consisting of L-lysine,L-valine, L-threonine, L-alanine or L-tryptophan. They include vitaminschosen from the group consisting of vitamin B1 (thiamine), vitamin B2(riboflavin),vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine),vitamin B12 (cyanocobalamin), nicotinic acid/nicotinamide and vitamin E(tocopherol). They also include organic acids which carry one to threecarboxyl groups, such as acetic acid, lactic acid, citric acid, malicacid or fumaric acid. Finally, they also include sugars, for example,trehalose. These compounds are optionally desired if they improve thenutritional value of the product.

These organic substances, including L-methionine and/or D-methionineand/or the racemic mixture D,L-methionine, can also be added, dependingon requirements, as a concentrate or pure substance in solid or liquidform during a suitable process step. These organic substances mentionedcan be added individually or as mixtures to the resulting orconcentrated fermentation broth, or also during the drying orgranulation process. It is likewise possible to add an organic substanceor a mixture of several organic substances to the fermentation broth anda further organic substance or a further mixture of several organicsubstances during a later process step, for example granulation.

The product described above is suitable as a feedstuffs additive, i.e.feed additive, for animal nutrition.

The L-methionine content of the animal feedstuffs additive isconventionally 1 wt. % to 80 wt. %, preferably 2 wt. % to 80 wt. %,particularly preferably 4 wt. % to 80 wt. %, and very particularlypreferably 8 wt. % to 80 wt. %, based on the dry weight of the animalfeedstuffs additive. Contents of 1 wt. % to 60 wt. %, 2 wt. % to 60 wt.%, 4 wt. % to 60 wt. %, 6 wt. % to 60 wt. %, 1 wt. % to 40 wt. %, 2 wt.% to 40 wt. % or 4 wt. % to 40 wt. % are likewise possible. The watercontent of the feedstuffs additive is conventionally up to 5 wt. %,preferably up to 4 wt. %, and particularly preferably less than 2 wt. %.

The invention also provides a process for the preparation of anL-methionine-containing animal feedstuffs additive from fermentationbroths, which comprises the steps

-   -   a) culture and fermentation of an L-methionine-producing        microorganism in a fermentation medium;    -   b) removal of water from the L-methionine-containing        fermentation broth (concentration);    -   c) removal of an amount of 0 to 100 wt. % of the biomass formed        during the fermentation; and    -   d) drying of the fermentation broth obtained according to a)        and/or b) to obtain the animal feedstuffs additive in the        desired powder or granule form.

If desired, one or more of the following steps can furthermore becarried out in the process according to the invention:

-   -   e) addition of one or more organic substances, including        L-methionine and/or D-methionine and/or the racemic mixture        D,L-methionine, to the products obtained according to a), b)        and/or c);    -   f) addition of auxiliary substances chosen from the group        consisting of silicas, silicates, stearates, grits and bran to        the substances obtained according to a) to d) for stabilization        and to increase the storability; or    -   g) conversion of the substances obtained according to a) to e)        into a form stable to the animal stomach, in particular rumen,        by coating with film-forming agents.

The analysis of L-methionine can be carried out by ion exchangechromatography with subsequent ninhydrin derivation, as described bySpackman et al. (Analytical Chemistry, 30, (1958) , 1190).

The process according to the invention is used for the fermentativepreparation of amino acids, in particular L-methionine.

The following microorganism was deposited as a pure culture on 14th Jun.2001 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen(DSMZ=German Collection of Microorganisms and Cell Cultures,Braunschweig, Germany) in accordance with the Budapest Treaty:

-   -   Escherichia coli DH5αmcr/pCREmetH as DSM 14354.

The present invention is explained in more detail in the following withthe aid of embodiment examples.

EXAMPLE 1

Preparation of a Genomic Cosmid Gene Library from Corynebacteriumglutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolatedas described by Tauch et al. (1995, Plasmid 33:168-179) and partlycleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). TheDNA fragments were dephosphorylated with shrimp alkaline phosphatase(Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP,Code no. 1758250).

The DNA of the cosmid vector SuperCosl (Wahl et al. (1987) Proceedingsof the National Academy of Sciences USA 84:2160-2164), obtained fromStratagene (La Jolla, USA, Product Description SuperCos1 Cosmid VectorKit, Code no. 251301) was cleaved with the restriction enzyme XbaI(Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Codeno. 27-0948-02) and likewise dephosphorylated with shrimp alkalinephosphatase. The cosmid DNA was then cleaved with the restriction enzymeBamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI,Code no. 27-0868-04). The cosmid DNA treated in this manner was mixedwith the treated ATCC13032 DNA and the batch was treated with T4 DNAligase (Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no.27-0870-04). The ligation mixture was then packedin phages with the aid of Gigapack II XL Packing Extract (Stratagene, LaJolla, USA, Product Description Gigapack II XL Packing Extract, Code no.200217).

For infection of the E. coli strain NM554 (Raleigh et al. 1988, NucleicAcid Research 16:1563-1575) the cells were taken up in 10 mM MgSO₄ andmixed with an aliquot of the phage suspension. The infection andtitering of the cosmid library were carried out as described by Sambrooket al. (1989, Molecular Cloning: A laboratory Manual, Cold SpringHarbor), the cells being plated out on LB agar (Lennox, 1955, Virology,1:190) with 100 mg/l ampicillin. After incubation overnight at 37° C.,recombinant individual clones were selected.

EXAMPLE 2

Isolation and Sequencing of the metH Gene

The cosmid DNA of an individual colony was isolated with the QiaprepSpin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) inaccordance with the manufacturer's instructions and partly cleaved withthe restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,Product Description Sau3AI, Product No. 27-0913-02). The DNA fragmentswere dephosphorylated with shrimp alkaline phosphatase (RocheDiagnostics GmbH, Mannheim, Germany, Product Description SAP, ProductNo. 1758250). After separation by gel electrophoresis, the cosmidfragments in the size range of 1500 to 2000 bp were isolated with theQiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

The DNA of the sequencing vector pZero-1, obtained from Invitrogen(Groningen, The Netherlands, Product Description Zero Background CloningKit, Product No. K2500-01) was cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI,Product No. 27-0868-04). The ligation of the cosmid fragments in thesequencing vector pZero-1 was carried out as described by Sambrook etal. (1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor),the DNA mixture being incubated overnight with T4 ligase (PharmaciaBiotech, Freiburg, Germany). This ligation mixture was thenelectroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7)into the E. coli strain DH5αmcr (Grant, 1990, Proceedings of theNational Academy of Sciences U.S.A., 87:4645-4649) and plated out on LBagar (Lennox, 1955, Virology, 1:190) with 50 mg/l zeocin.

The plasmid preparation of the recombinant clones was carried out withBiorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany) . Thesequencing was carried out by the dideoxy chain termination method ofSanger et al. (1977, Proceedings of the National Academy of SciencesU.S.A., 74:5463-5467) with modifications according to Zimmermann et al.(1990, Nucleic Acids Research, 18:1067). The “RR dRhodamin TerminatorCycle Sequencing Kit” from PE Applied Biosystems (Product No. 403044,Weiterstadt, Germany) was used. The separation by gel electrophoresisand analysis of the sequencing reaction were carried out in a“Rotiphoresis NF Acrylamide/Bisacrylamide” Gel (29:1) (Product No.A124.1, Roth, Karlsruhe, Germany) with the “ABI Prism 377” sequencerfrom PE Applied Biosystems (Weiterstadt, Germany).

The raw sequence data obtained were then processed using the Stadenprogram package (1986, Nucleic Acids Research, 14:217-231) version 97-0.The individual sequences of the pZero1 derivatives were assembled to acontinuous contig. The computer-assisted coding region analysis wasprepared with the XNIP program (Staden, 1986, Nucleic Acids Research,14:217-231).

The resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis ofthe nucleotide sequence showed an open reading frame of 3662 base pairs,which was called the metH gene. The metH gene codes for a protein of1221 amino acids.

EXAMPLE 3

Preparation of the Strain C. glutamicum ATCC13032/pCREmetH

3.1 Amplification of the metH Gene

From the strain ATCC13032, chromosomal DNA was isolated by the method ofEikmanns et al. (Microbiology 140: 1817-1828 (1994)). Starting from thenucleotide sequences of the methionine biosynthesis genes metH (SEQ IDNo. 1) of C. glutamicum ATCC13032, the following oligonucleotides werechosen for the polymerase chain reaction (PCR) (see SEQ ID No. 3 and SEQID No. 4): metH-EVP5:5′-GATCTAAGATCTAAAGGAGGACAACCATGTCTACTTCAGTTACTTCACCAGC-3′ metH-EVP3:5′-GATCTAGTCGACCCCTCTCAAAGGTGTTAGAC-3′

The primers shown were synthesized by MWG-Biotech AG (Ebersberg,Germany) and the PCR reaction was carried out by the standard PCR methodof Innis et al. (PCR Protocols. A Guide to Methods and Applications,1990, Academic Press) with Pwo-Polymerase from Roche Diagnostics GmbH(Mannheim, Germany). With the aid of the polymerase chain reaction, theprimers allow amplification of a DNA fragment 3718 bp in size, whichcarries the metH gene.

Furthermore, the primer metH-EVP5 contains the sequence for the cleavagesite of the restriction endonuclease BglII and the primer metH-EVP3 thecleavage site of the restriction endonuclease SalI, which are marked byunderlining in the nucleotide sequence shown above.

The metH fragment 3718 bp in size was cleaved with the restrictionendonucleases BglII and SalI. The batch was separated by gelelectrophoresis and the metH fragment (approx. 3700 bp) was thenisolated from the agarose gel with the QiaExII Gel Extraction Kit(Product No. 20021, Qiagen, Hilden, Germany).

3.2 Cloning of metH in the Vector pZ8-1

The E. coli -C. glutamicum shuttle expression vector pZ8-1 (EP 0 375889) was used as the base vector for the expression.

DNA of the plasmid pZ8-1 was cleaved completely with the restrictionenzymes BamHI and SalI and then dephosphorylated with shrimp alkalinephosphatase (Roche Diagnostics GmbH, Mannheim, Germany, ProductDescription SAP, Product No. 1758250).

The metH fragment approx. 3700 bp in size isolated from the agarose gelin example 3.1 and cleaved with the restriction endonucleases BglII andSalI was mixed with the vector pZ8-1 prepared in this way and the batchwas treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany,Product Description T4-DNA-Ligase, Code no.27-0870-04).

The ligation batch was transformed in the E. coli strain DH5αmcr(Hanahan, In: DNA cloning. A Practical Approach. Vol. I. IRL-Press,Oxford, Washington DC, U.S.A.). Selection of plasmid-carrying cells wasmade by plating out the transformation batch on LB agar (Lennox, 1955,Virology, 1:190) with 50 mg/l kanamycin. After incubation overnight at37° C., recombinant individual clones were selected. Plasmid DNA wasisolated from a transformant with the Qiaprep Spin Miniprep Kit (ProductNo. 27106, Qiagen, Hilden, Germany) in accordance with themanufacturer's instructions and checked by restriction cleavage. Theresulting plasmid was called pCREmetH. The strain E. coliDH5αmcr/pCREmetH was deposited as a pure culture on 14th Jun. 2001 atthe Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ=GermanCollection of Microorganisms and Cell Cultures, Braunschweig, Germany)in accordance with the Budapest Treaty as DSM 14354.

3.3 Preparation of the Strain C. glutamicum ATCC13032/pCREmetH

The vector pCREmetH obtained in example 3.2 was electroporated in thestrain C. glutamicum ATCC13032 using the electroporation methoddescribed by Liebl et al. (FEMS Microbiology Letters, 53:299-303(1989)). Selection of the plasmid-carrying cells took place on LBHISagar comprising 18.5 g/l brain-heart infusion broth, 0.5 M sorbitol, 5g/l Bacto-tryptone, 2.5 g/l Bacto-yeast extract, 5 g/l NaCl and 18 g/lBacto-agar, which had been supplemented with 25 mg/l kanamycin.Incubation was carried out for 2 days at 33° C.

Plasmid DNA was isolated from a transformant by conventional methods(Peters-Wendisch et al., 1998, Microbiology 144, 915-927) and checked byrestriction cleavage. The resulting strain was called ATCC13032pCREmetH.

EXAMPLE 4

Preparation of Methionine with the strain C. glutamicumATCC13032/pCREmetH

The C. glutamicum strain ATCC13032/pCREmetH obtained in example 3 wascultured in a nutrient medium suitable for the production of methionineand the methionine content in the culture supernatant was determined.

For this, the strain was first incubated on an agar plate with thecorresponding antibiotic (brain-heart agar with kanamycin (25 mg/l)) for24 hours at 33° C. Starting from this agar plate culture, a preculturewas seeded (10 ml medium in a 100 ml conical flask). The medium MM wasused as the medium for the preculture. Medium MM CSL (corn steep liquor)5 g/l MOPS (morpholinopropanesulfonic acid) 20 g/l Glucose (autoclavedseparately) 50 g/l Salts: (NH₄)₂SO₄ 25 g/l KH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O1.0 g/l CaCl₂ * 2 H₂O 10 mg/l FeSO₄ * 7 H₂O 10 mg/l MnSO₄ * H₂O 5.0 mg/lBiotin (sterile-filtered) 0.01 mg/l Vitamin B12 (sterile-filtered) 0.02mg/l Thiamine * HCl (sterile-filtered) 0.2 mg/l CaCO₃ 25 g/l

The CSL, MOPS and the salt solution were brought to pH 7 with aqueousammonia and autoclaved. The sterile substrate and vitamin solutions werethen added, as well as the CaCO₃ autoclaved in the dry state.

Kanamycin (25 mg/l) was added to this. The preculture was incubated for16 hours at 33° C. at 240 rpm on a shaking machine. A main culture wasseeded from this preculture such that the initial OD (660 nm) of themain culture was 0.1. Medium MM was also used for the main culture.

Culturing is carried out in a 10 ml volume in a 100 ml conical flaskwith baffles. Kanamycin (25 mg/l) was added. Culturing was carried outat 33° C. and 80% atmospheric humidity.

After 72 hours, the OD was determined at a measurement wavelength of 660nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount ofmethionine formed was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivation with ninhydrin detection.

The result of the experiment is shown in table 1. TABLE 1 OD MethionineStrain (660 nm) mg/l ATCC13032 12.3 1.4 ATCC13032/pCREmetH 14.3 5.3

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1: Plasmid pCREmetH

The abbreviations used in the figures have the following meaning: Km:Resistance gene for kanamycin metH: metH gene of C. glutamicum Ptac: tacpromoter T1 T2: Terminator T1T2 of the rrnB gene of E. coli rep:Plasmid-coded replication origin for C. glutamicum (of pHM1519) EcoRI:Cleavage site of the restriction enzyme EcoRI SalI: Cleavage site of therestriction enzyme SalI

This disclosure is based on priority documents DE 100 38 050.6, DE 10109 687.9 and U.S. Pat. No. 60/294,251, each incorporated by reference.

Obviously, numerous modifications of the invention are possible in viewof the above teachings. Therefore, within the scope of the appendedclaims, the invention may be practiced otherwise than as specificallydescribed herein.

1-34. (canceled)
 35. A nucleic acid comprising at least 15 consecutivenucleotides of a polynucleotide encoding SEQ ID NO: 2 or its fullcomplement, and which binds to a nucleic acid which encodes apolypeptide having homocysteine methyl transferase activity.
 36. Thenucleic acid of claim 35, which comprises at least 20 consecutivenucleotides of a polynucleotide encoding SEQ ID NO: 2 or its fullcomplement, and which binds to a nucleic acid which encodes apolypeptide having homocysteine methyl transferase activity.
 37. Thenucleic acid of claim 35, which comprises at least 30 consecutivenucleotides of a polynucleotide encoding SEQ ID NO: 2 or its fullcomplement, and which binds to a nucleic acid which encodes apolypeptide having homocysteine methyl transferase activity.
 38. Thenucleic acid of claim 35, which comprises at least 50 consecutivenucleotides of a polynucleotide encoding SEQ ID NO: 2 or its fullcomplement, and which binds to a nucleic acid which encodes apolypeptide having homocysteine methyl transferase activity.
 39. Thenucleic acid of claim 35 which comprises at least 15 consecutivenucleotides of a polynucleotide encoding SEQ ID NO:
 2. 40. The nucleicacid of claim 35 which comprises at least 15 consecutive nucleotides ofthe full complement of a polynucleotide encoding SEQ ID NO:
 2. 41. Amethod of identifying a nucleic acid which encodes a polypeptide havinghomocysteine methyltransferase II activity, comprising probing a nucleicacid sample with the nucleic acid of claim
 35. 42. The method of claim41, wherein said nucleic acid sample comprises RNA, DNA or cDNA.
 43. Amethod for producing a nucleic acid which encodes a polypeptide havinghomocysteine methyltransferase II activity, comprising priming a nucleicacid sample with the nucleic acid of claim
 35. 44. The method of claim43, wherein said nucleic acid sample comprises RNA, DNA or cDNA.
 45. Aprocess for preparing an L-methionine-containing animal feedstuffsadditive, comprising: a) culturing an L-methionine-producingmicroorganism in a fermentation medium to produce a fermented mediumcontaining L-methionine, b) removing water from the fermented medium(concentration); c) removing 0 to 100 wt. % of the biomass from thefermented medium; and/or d) drying the fermented medium, optionally toform a powder or granules.
 46. The process of claim 45, wherein theexpression of at least one gene in the biosynthesis pathway forL-methionine in said microorganism is enhanced.
 47. The process of claim46, wherein said gene is selected from the group consisting of: the lysCgene which codes for a feed back resistant aspartate kinase, the gapgene which codes for glycerolaldehyde 3-phosphate dehydrogenase, the pgkgene which codes for 3-phosphoglycerate kinase, the pyc gene which codesfor pyruvate carboxylase, the tpi gene which codes for triose phosphateisomerase the metA gene which codes for homoserine 0-acetyltransferasethe metB gene which codes for cystathionine gamma-synthase aecD genewhich codes for cystathionine gamma-lyase glyA gene which codes forserine hydroxymethyltransferase and metY gene which codes for0-acetylhomoserine-sulfhydrylase.
 48. The process of claim 45, whereinthe expression of at least one gene which decreases L-methionine levelsin said microorganism is reduced or eliminated.
 49. The process of claim48, wherein said gene is selected from the group consisting of: the thrBgene which codes for homoserine kinase the ilvA gene which codes forthreonine dehydratase the thrC gene which codes for threonine synthasethe ddh gene which codes for meso-diaminopimelate D-dehydrogenase thepck gene which codes for phosphoenol pyruvate carboxykinase the pgi genewhich codes for glucose 6-phosphate isomerase and the poxB gene whichcodes for pyruvate oxidase.
 50. The process of claim 45, whereinexpression of the polynucleotide encoding the metF gene product isenhanced.
 51. The process of claim 45, wherein microorganisms of thespecies Corynebacterium glutamicum are employed.
 52. The process ofclaim 51, wherein the Corynebacterium glutamicum strainATCC13032/pCREmetF is employed.
 53. The process of claim 45, furthercomprising at least one of the following steps: e) adding at least oneorganic substance to the product obtained by steps b), c) and/or d); f)adding at least one auxiliary substance selected from the groupconsisting of silicas, silicates, stearates, grits and bran to theproduct obtained by steps to b), c), d) and/or e); and/or g) coating theproduct obtained by steps b), c), d), e) and/or f) with a film-formingagent.
 54. The process of claim 53, wherein the film-forming agent is atleast one selected from the group consisting of metal carbonates,silicas, silicates, alginates, stearates, starches, gums and celluloseethers.
 55. The process of claim 45, wherein a portion of the biomass isremoved.
 56. The process of claim 45, wherein essentially 100% of thebiomass is removed.
 57. The process as claimed in claim 45, wherein thewater content in said feedstuff additive is up to 5 wt. %.
 58. Theprocess of claim 45, wherein the water content in said feedstuffadditive is less than 2 wt. %.
 59. An animal feedstuffs additiveprepared by the process of claim
 45. 60. The animal feedstuffs additiveof claim 59, which comprises 1 wt. % to 80 wt. % L-methionine,D-methionine, D,L-methionine or a mixture thereof, based on its dryweight.